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Gregg Randolph

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16S0C4 and 16S0D4 were used.

...

  1. Pull aliquots of some high read count and low read count samples. (Pending new low read samples).

  2. Quantify all samples not including blanks or mock community.

  3. For previously low yielding samples, repeat at typical template amount, and at 2x, and 4x volume added to the reaction (in column 2,3,4, and 6, 7, 8). 6x8 samples in low yield, 8 in high yield, 8 of mock community, 8 of blank (ISD only). 72 reactions with different templates. See template below for clarification.

  4. Template layout is replicated/duplicated, with different barcodes for duplicates.

  5. 1 set of 36 products will not be adjusted for pooling.

  6. 1 set of 36 will be adjusted for pooling per qPCR results.

Start Reaction Plate Setup:

...

Red samples (bottom half) will either just be pooled per qPCR numbers or concentrated via SpeedVac, reconstituted to a higher concentration, and pooled by qPCR results. (Pending method instructions).

“ul for pooling” was calculated by multiplying the lowest molarity (3.15) times the maximum V available (35 ul) to get ~110. This result was then divided by each sample’s molarity to obtain the ul needed for equimolar pooling rounded to one decimal place.

...

Well Position

Sample

qPCR result

ul for pooling

Repeated qPCR Result

qPCR Duplicated Run Results 02/09

1E

SAG192203_R

54.32

2.0

1.58

2.04

2E

SAG_S30P2R_R_2uL

54.14

2.0

1.22

1.16

3E

SAG_S30P2R_R_4uL

54.03

2.0

2.05

1.34

4E

SAG_S30P2R_R_8uL

11.34

9.7

5.70E-01

3.61E-01

5E

Mock_Comm_R

65.14

1.7

2.07

1.62

6E

SAG192199_R_2uL

71.08

1.5

2.09

1.25

7E

SAG192199_R_4uL

95.41

1.2

2.34

3.91

8E

SAG192199_R_8uL

80.56

1.4

2.65

6.07

9E

Blank_ISD_R

19.23

5.7

1.8

2.25

1F

SAG191508_R

64.52

1.7

31.69

37.26

2F

SAG_S38P1R_R_2uL

60.01

1.8

1.51

1.56

3F

SAG_S38P1R_R_4uL

56.04

2.0

6.72

5.75

4F

SAG_S38P1R_R_8uL

3.15

34.9

3.86E-01

5F1.42E-01

5F

Mock_Comm_R

63.52

1.7

2.51

2.11

6F

SAG192077_R_2uL

65.88

1.7

2.38

1.58

7F

SAG192077_R_4uL

84.27

1.3

2.37

3.1

8F

SAG192077_R_8uL

83.59

1.3

3.03

4.24

9F

Blank_ISD_R

13.12

8.4

7.78E-01

9.15E-01

1G

SAG_S16P3R_R

26.49

4.2

11.34

14.1

2G

SAG_S15P4R_R_2uL

53.51

2.1

14.31

16.26

3G

SAG_S15P4R_R_4uL

26.2

4.2

1.33

1.56

4G

SAG_S15P4R_R_8uL

12.91

8.5

6.56E-01

6.53E-01

5G

Mock_Comm_R

79.7

1.4

1.9

2.14

6G

SAG190271_R_2uL

70.2

1.6

1.59

1.75

7G

SAG190271_R_4uL

62.73

1.8

1.81

1.95

8G

SAG190271_R_8uL

59.32

1.9

6.33E-01

2.61

9G

Blank_ISD_R

20.04

5.5

6.79E-01

1.13

1H

SAG_S44P2R_R

19.92

5.5

2.4

4.46

2H

SAG190005_R_2uL

54.03

2.0

2.89

6.26

3H

SAG190005_R_4uL

57.05

1.9

2.72

3.62

4H

SAG190005_R_8uL

60.28

1.8

4.37

3.46

5H

Mock_Comm_R

64.83

1.7

2.17

2.28

6H

SAG190238_R_2uL

68.19

1.6

76.62

62

7H

SAG190238_R_4uL

55.07

2.0

2.16

1.83

8H

SAG190238_R_8uL

41.35

2.7

2.5

3.88

9H

Blank_ISD_R

18.32

6.0

5.57E-01

1.36

qPCR blue/red pools:

  • Make 1:1000 dilutions of blue and red pools by adding 1 ul to 999 ul TE in a 1.5mL tubes.

  • Run each pool in triplicate.

  • Add 16 ul of Illumina Library Quantification MasterMix to each well:

...

Use R to associate NS2 input concentrations with sample names, read counts, and % ISD reads

Repeat qPCR post calibration:

qPCR MMX

  • Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn

Reagent

# of rxns

ul needed

10 ul

KAPA SYBR FAST qPCR MM (2X)

100

1000

2 ul

Primer Premix (10X)

100

200

4 ul

Ultra Pure Water

100

400

16 ul

Total Volume

100

1600

  • Add 4 ul of template, pool, or standards to each well:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

SAG192203

Tube 127

2ul SAG_S30P2R

PLT2: G6

4ul SAG_S30P2R

PLT2: G6

8ul SAG_S30P2R

PLT2: G6

mock community

2ul SAG192199

Tube 123

4ul SAG192199

Tube 123

8ul SAG192199

Tube 123

 blank with ISD only

NTC

NTC

NTC

B

SAG191508

Tube 73

2ul SAG_S38P1R

PLT2: B9

4ul SAG_S38P1R

PLT2: B9

8ul SAG_S38P1R

PLT2: B9

mock community

2ul SAG192077

Tube 96

4ul SAG192077

Tube 96

8ul SAG192077

Tube 96

blank with ISD only

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

SAG_S16P3R

PLT1: G3

2ul SAG_S15P4R

PLT2: B2

4ul SAG_S15P4R

PLT2: B2

8ul SAG_S15P4R

PLT2: B2

mock community

2ul SAG190271

Tube 258

4ul SAG190271

Tube 258

8ul SAG190271

Tube 258

  blank with ISD only

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

SAG_S44P2R

PLT1: F7

2ul SAG190005

Tube 33

4ul SAG190005

Tube 33

8ul SAG190005

Tube 33

mock community

2ul SAG190238

Tube 255

4ul SAG190238

Tube 255

8ul SAG190238

Tube 255

  blank with ISD only

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

SAG192203

Tube 127

2ul SAG_S30P2R

PLT2: G6

4ul SAG_S30P2R

PLT2: G6

8ul SAG_S30P2R

PLT2: G6

mock community

2ul SAG192199

Tube 123

4ul SAG192199

Tube 123

8ul SAG192199

Tube 123

 blank with ISD only

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

SAG191508

Tube 73

2ul SAG_S38P1R

PLT2: B9

4ul SAG_S38P1R

PLT2: B9

8ul SAG_S38P1R

PLT2: B9

mock community

2ul SAG192077

Tube 96

4ul SAG192077

Tube 96

8ul SAG192077

Tube 96

blank with ISD only

2 pM Std

2 pM Std

2 pM Std

G

SAG_S16P3R

PLT1: G3

2ul SAG_S15P4R

PLT2: B2

4ul SAG_S15P4R

PLT2: B2

8ul SAG_S15P4R

PLT2: B2

mock community

2ul SAG190271

Tube 258

4ul SAG190271

Tube 258

8ul SAG190271

Tube 258

  blank with ISD only

20 pM Std

20 pM Std

20 pM Std

H

SAG_S44P2R

PLT1: F7

2ul SAG190005

Tube 33

4ul SAG190005

Tube 33

8ul SAG190005

Tube 33

mock community

2ul SAG190238

Tube 255

4ul SAG190238

Tube 255

8ul SAG190238

Tube 255

  blank with ISD only

 

 

 

Results 02/08/2022:

View file
nameLR_II_FEB22.csv

Results for duplicate qPCR run on 02/09/2022:

View file
nameLR_II_FEB22_2.csv

Results look the same from the 02/08 run.